The authors have declared that no competing interests exist.
Conceived and designed the experiments: Yan Zhang WBX. Performed the experiments: Yan Zhang XJT ALC NYM STX ZZ JHZ YPZ XJW XYH SLZ Yong Zhang WT HL. Analyzed the data: Yan Zhang JS. Contributed reagents/materials/analysis tools: Yan Zhang YPZ XJW XYH WBX. Wrote the paper: Yan Zhang WBX.
Genetic recombination is a well-known phenomenon for enteroviruses. To investigate the genetic characterization and the potential recombination of enterovirus 71 (EV71) circulating in China, we determined the 16 complete genome sequences of EV71 isolated from Hand Foot Mouth Disease (HFMD) patients during the large scale outbreak and non-outbreak years since 1998 in China. The full length genome sequences of 16 Chinese EV71 in present study were aligned with 186 genome sequences of EV71 available from GenBank, including 104 China mainland and 82 international sequences, covering the time period of 1970–2011. The oldest strains of each subgenotype of EV71 and prototype strains of HEV-A were included to do the phylogenetic and Simplot analysis. Phylogenetic analysis indicated that all Chinese strains were clustered into C4 subgenotype of EV71, except for HuB/CHN/2009 clustered into A and Xiamen/CHN/2009 clustered into B5 subgenotype. Most of C4 EV71 were clustered into 2 predominant evolutionary branches: C4b and C4a evolutionary brunches. Our comprehensive recombination analysis showed the evidence of genome recombination of subgenotype C4 (including C4a and C4b) sequences between structural genes from genotype C EV71 and non-structural genes from the prototype strains of CAV16, 14 and 4, but the evidence of intratypic recombination between C4 strains and B subgenotype was not enough strong. This intertypic recombination C4 viruses were first seen in 1998 and became the predominant endemic viruses circulating in China mainland for at least 14 years. A shift between C4a and C4b evolutionary brunches of C4 recombination viruses were observed, and C4a viruses have been associated with large scale nationwide HFMD outbreak with higher morbidity and mortality since 2007.
The Enterovirus genus in the family Picornaviridae consists of 4 species with strains isolated from humans: Human enterovirus A (HEV-A), HEV-B, HEV-C, HEV-D
EV71 infection, which was first reported in the USA, has been a recurrent feature in the Asia-Pacific region since its first outbreak in Sarawak, Malaysia in 1997
In China, large scale EV71 outbreak of HFMD associated with acute neurological disease occurred in 2007 at Linyi City, Shandong province
Genomic recombinations are well known to contribute to genetic variations and evolution of enteroviruses. Complete genome analysis of prototype HEV-A indicated that recombination in the nonstructural region has played a role in the evolution of some HEV-A prototypes
Our previous studies have been confirmed that the large-scale HFMD outbreaks with fatal neurological complications that have occurred since 2008 are mainly due to subgenotype C4a of HEV71, which was identified as a recombination virus with CVA16 in 3D region
The intensive surveillance for HEV71 circulation maintained by mainland China during and after the 2007 outbreak permitted a detailed analysis of a large number of isolates from the HFMD patients by complete genome analysis methods. To investigate the genetic characterization of complete genome of C4 subgenotype EV71 strains and the recombination with prototype strains of HEV-A strains, we performed a large scale genomic sequence analysis of isolates (n = 202) collected from 17 countries worldwide over a 4 decades period. We sequenced and analyzed the entire genome sequences of 16 C4 subgenotype EV71 isolated from HFMD or encephalomyelitis or fatal patients during a period of both before and after EV71 large-scale outbreak in mainland China. The phylogenetic analysis, similarity plot and bootscan analysis were performed to analyze the phylogenetic relationship and potential recombination between C4 subgenotype EV71 strain circulating in mainland China, oldest strain of EV71 subgenotype and other prototype stains of HEV-A species.
The full length genome sequences of 16 China EV71 in present study were aligned with 186 genome sequences of EV71 available from GenBank, including 104 China mainland and 82 international sequences from South and East Asia(22), Australia(8), and Europe(8), America (10), Japan(9), Korea(2), Taiwan(23), and the collection date of the clinical specimens of 186 sequences covered from 1970–2011(
Red dots indicate the sequences from Mainland China and blue triangles indicate the sequences downloaded from GenBank. The prototype of EV71(BrCr), CVA-16(G-10) and the oldest sequences of different subgenotypes of EV71(B0-5, C1-5 subgenotypes) were included as well. Green color strain name indicate the mild case, purple color strain name indicate the severe case and pink color strain name indicate the fatal case.
A comprehensive comparison of nucleotide acid and deduced amino acid sequence identities between C4a, C4b and the oldest strain of each subgenotype of EV71 and other HEV-A species prototype strains is shown in
Abbreviation | Strain name | Genbank Accession Number | The place of isolation | The year of isolation | Genotype/Subgenotype | prototype or representative strains |
EV71 | 10857/NED/1966 | AB575912 | Netherlands | 1966 | B0 | oldest |
EV71 | 11977/NED/1971 | AB575913 | Netherlands | 1971 | B1 | oldest |
EV71 | 20233/NED/1983 | AB575923 | Netherlands | 1983 | B2 | oldest |
EV71 | MY821-3/1997 | DQ341367 | Singapore | 1997 | B3 | oldest |
EV71 | 5865/sin/000009/SIN/2000 | AF316321 | Singapore | 2000 | B4 | oldest |
EV71 | 5511-SIN-00 | DQ341364 | Singapore | 2000 | B5 | oldest |
EV71 | NED/1991 | AB575935 | Netherlands | 1991 | C1 | oldest |
EV71 | Tainan/5746/98/TW/1998 | AF304457 | Taiwan | 1998 | C2 | oldest |
EV71 | 06-KOR-00/KOR/2000 | DQ341355 | South Korea | 2000 | C3 | oldest |
EV71 | SHZH98/CHN/1998 | AF302996 | China | 1998 | C4 | oldest |
EV71 | 2007-07364/TW/2007 | EU527983 | Taiwan | 2007 | C5 | oldest |
EV71 | BrCr/USA/1970 | U22521 | USA | 1970 | A | prototype |
COXA2 | Fleetwood/USA/1947 | AY421760 | USA | 1947 | A2 | prototype |
COXA3 | Olson/USA/1948 | AY421761 | USA | 1948 | A3 | prototype |
COXA4 | High Point/USA/1948 | AY421762 | USA | 1948 | A4 | prototype |
COXA5 | Swartz/USA/1950 | AY421763 | USA | 1950 | A5 | prototype |
COXA6 | Gdula/USA/1949 | AY421764 | USA | 1949 | A6 | prototype |
COXA7 | Parker/USA/1949 | AY421765 | USA | 1949 | A7 | prototype |
COXA8 | Donovan/USA/1949 | AY421766 | USA | 1949 | A8 | prototype |
COXA10 | Kowalik/USA/1950 | AY421767 | USA | 1950 | A10 | prototype |
COXA12 | Texas-12/USA/1948 | AY421768 | USA | 1948 | A12 | prototype |
COXA14 | G-14/SOA/1950 | AY421769 | Republic of South Africa | 1950 | A14 | prototype |
COXA16 | G10/SOA/1951 | U05876 | Republic of South Africa | 1951 | A16 | prototype |
Region | %identity | |||||||||||
C4a | C4b | |||||||||||
BrCr | Other EV71 |
CA16 | CA14 | CA4 | Other HEV-A | BrCr | Other EV71 |
CA16 | CA14 | CA4 | Other HEV-A | |
5′UTRa | 86.7 | 85.7–88.7 | 85.0 | 87.0 | 86.8 | 74.9–88.3 | 87.1 | 87.0–89.5 | 85.7 | 87.3 | 87.5 | 74.9–88.6 |
P1 | 73.2 | 73.7–86.9 | 48.8 | 40.6 | 41.1 | 35.3–38.2 | 73.0 | 73.3–87.9 | 47.8 | 40.6 | 39.0 | 33.9–37.2 |
VP4 | 100.0 | 85.5–100.0 | 75.5 | 52.7 | 61.6 | 57.3–63.7 | 100.0 | 85.5–100.0 | 75.5 | 52.7 | 61.6 | 57.3–63.7 |
VP2 | 64.5 | 66.0–78.4 | 41.5 | 45.9 | 45.9 | 31.2–49.2 | 62.4 | 59.5–83.8 | 37.9 | 47.6 | 45.0 | 30.2–47.6 |
VP3 | 89.2 | 88.1–93.2 | 77.6 | 66.4 | 66.4 | 59.7–69.0 | 90.2 | 89.7–94.2 | 78.7 | 67.1 | 65.1 | 58.3–69.0 |
VP1 | 73.2 | 73.7–92.0 | 48.8 | 40.6 | 41.1 | 35.3–41.6 | 73.0 | 73.3–87.9 | 47.8 | 40.6 | 39.0 | 33.9–39.6 |
P2 | 95.4 | 95.2–98.1 | 97.4 | 97.0 | 96.5 | 76.2–96.8 | 96.1 | 95.6–98.4 | 97.7 | 97.7 | 97.5 | 76.7–97.4 |
2A | 94.5 | 93.8–98.0 | 95.9 | 95.2 | 94.5 | 62.4–95.2 | 95.9 | 94.5–98.0 | 95.9 | 96.6 | 95.9 | 63.4–96.6 |
2B | 93.7 | 90.5–98.0 | 98.0 | 98.0 | 96.9 | 70.9–96.9 | 94.8 | 91.6–99.0 | 99.0 | 99.0 | 98.0 | 77.2–98.0 |
2C | 96.3 | 94.7–98.2 | 97.8 | 97.5 | 97.2 | 82.7–97.8 | 96.6 | 99.1–95.0 | 98.2 | 97.8 | 98.2 | 82.4–98.2 |
P3 | 92.5 | 92.7–97.1 | 97.5 | 97.3 | 97.1 | 79.5–94.8 | 92.0 | 92.5–97.5 | 97.4 | 97.7 | 97.1 | 79.0–94.5 |
3A | 97.6 | 91.3–98.8 | 97.6 | 98.8 | 98.8 | 64.7–98.8 | 96.4 | 90.0–97.6 | 96.4 | 97.6 | 97.6 | 63.0–97.6 |
3B | 31.6 | 31.6–73.7 | 47.4 | 57.9 | 63.2 | 21.1–47.4 | 31.6 | 36.8–78.9 | 63.2 | 78.9 | 84.2 | 31.6–57.9 |
3C | 92.5 | 91.9–97.7 | 97.2 | 97.7 | 97.7 | 80.3–96.0 | 92.5 | 93.1–98.9 | 98.3 | 98.9 | 98.9 | 80.3–96.0 |
3D | 91.8 | 92.7–96.9 | 98.0 | 97.1 | 96.9 | 81.5–94.4 | 91.3 | 92.3–97.3 | 97.6 | 97.6 | 96.7 | 81.5–93.9 |
3′UTRa | 67.6 | 64.7–76.5 | 70.6 | 76.5 | 79.4 | 29.4–70.6 | 67.6 | 61.8–64.7 | 70.6 | 70.6 | 73.5 | 29.4–70.6 |
not including the prototype strain of C4 subgenotype: SHZH98 a nucleotide acid sequence identities between Chinese representative strains of EV71 and other prototype strains of HEV-A species.
To investigate the genetic relationship between the Chinese C4 EV71 strains and the oldest strains of EV71 subgenotypes and other prototype HEV-A strains available in GenBank, the phylogenetic trees based on the full length of genome and 5′UTR, P1, P2, P3, 3′UTR region of the genome were constructed respectively (
The neighbour-joining trees were constructed from alignment of the 5' UTR (a), P1 (b), P2 (c), P3 (d) and 3′UTR (e) genomic region, respectively. The percentage of bootstrap (percentage of 1000 pseudoreplicate datasets) replicates supporting the trees are indicated at the nodes; for clarity, only values over 80% are shown. The branch lengths are proportional to the genetic distances corrected using Kimura-two-parameter substitution model.
If we performed the phylogenetic analysis based on the detailed coding region of P2 and P3, we found a slightly different phylogenetic relationship between C4 subgenotype and other subgenotype of EV71 and other HEV-A prototype strains (
The genome sequences of Chinese C4 subgenotype viruses and all available HEV-A prototype strains were analyzed with Simplot software, using the representative strain of each lineage in turn as the query sequence. Similarity plot analyses demonstrated that C4a, C4b viruses showed the highest degree of similarity to the C genotype of EV71 in the capsid region, but in the non-capsid region, C4a, C4b viruses were all contained an unidentified sequence in the P2 and P3 coding region that was apparently not related to those of EV71 strains (
Each analysis used each of the two lineages viruses as the query sequence. A sliding window of 1000 nucleotides moving in 20-nucleotide steps was used in this analysis. (a) C4b virus: SH-17/SH/CHN/2002; (b) C4a virus: HeN09-17/HeN/CHN/2009.
Subsequent bootscan analyses indicated possible recombination events. C4a, C4b EV71 strains were most closely related to the C genotype of EV71 in the 5′ half of the genome, which is consistent with the Simplot analysis results. However, after the junction sequences between VP1 and 2A, the bootscan graph exhibited a sound phylogenetic relationship between C4a, C4b EV71 strains and CVA-16, 14, 4.
Support for the inter-typic recombination involving 3 prototype strains of HEV-A and EV-71 were demonstrated by one major breakpoint identified in nucleotide 3700–3826 within the 2A gene of the complete genome (
4 orphan strains of C4 subgenotype, FJ194964-EV71/GDFS/3/2008, GQ994989-CQ/CHN/2009, FJ607337-SHZH/CHN/2008Fatal, HQ423143-km186/yYN/CHN/2009, were performed with Simplot analysis. A strain from the fatal case, FJ607337-SHZH/CHN/2008Fatal, shared the similar homology (>94%) with other 3 orphan strains from non-fatal cases in 5′UTR, P1 and P2 region of the genome, but the homology decreased gradually in the P3 region down to 85% in 3D region(not shown). It has been reported that sequences in the viral RNA-dependent RNA polymerase 3D (3Dpol) gene are important in determining the neurovirulence of polioviruses
C4 recombination virus was first isolated in ShenZhen, Guangdong province of China in 1998
For the geographical transmission of the recombination C4 viruses, we found that C4b viruses circulated in southern provinces of China, such as, Guangdong, Chongqing, Shanghai,Guangxi; and C4a viruses transmitted extensively throughout.
The phylogenetic analysis of complete genome of EV71 circulating in mainland China in this present study showed all Chinese strains were clustered into C4 subgenotype group, except for HuB/CHN/2009 clustered into A and Xiamen/CHN/2009 clustered into B5(
In this study, one B5 subgenotype virus was found in Xiamen (located in south of China) in 2009, which is the first B5 isolate found in China mainland. B5 subgenotype viruses were first found in Japan and Malaysia in 2003
No reported severe or fatal cases caused by C4b viruses occurred before large scale outbreak of HFMD in mainland China during 1993–2003 based on our analysis of complete genome. However, during the large scale outbreak of HFMD, increasing neurovirulence associated with C4a virus is a big concern for public health in mainland China. Our study on the analysis of complete genome, C4a viruses caused different phenotype of disease from mild to fatal. No specific lineages were associated with severe or fatal or mild cases. This indicated the viruses isolated from different phenotypic patients derived from the common ancestor and evolutes to different lineages by mutating gradually. We speculated that both viruses and host factor contributed to the phenotype of the disease.
Genomic recombinations are well known to contribute to genetic variations and evolution of enteroviruses. A range of enteroviruses of various serotypes or genotypes co-circulating in populations at some point of time was reported by different research groups
Interestingly, the EV-71 isolates of subgenotype B3 shared the similar recombination pattern with C4. These isolates had high sequence similarity to EV-71 genotype B, CV-A4, CV-A14 and CV-A16/G10 at P2 genomic region (≥81%) and high sequence similarity to CV-A4, CV-A14 and CV-A16 at P3 genomic region ((≥83%),
The clustering of isolates of subgenotype C4 with B3 and CVA4, 14, 16 at the 3' UTR genomic region was consistent with the previous clustering at the P2 and P3 genomic regions. No significant segregation (<30% bootstrap support), however, was observed for the remaining isolates and this was perhaps due to the short sequence length of the 3' UTR(∼83nt). Based on these results, it appeared that sequences of genes at the 3' half of the EV-71 genome contributed to the multiple and diverse EV71 subgenotypes and these genes showed high similarity to different HEV-A viruses.
The incongruent phylogenies and simplot similarity analyses imply that recombination has played an evident role in the evolution of C4 EV71 viruses. C4a and C4b clearly contained sequences in the non-capsid region that are also present in CVA4,14,16, suggesting that these three HEV-A strains and C 4 subgenotype viruses of EV71 have a shared evolutionary history, despite their lack of similarity in the capsid region. However, the exact recombination counterpart of HEV-A could not be found because there is not sufficient data regarding the P2 and P3 sequences of the HEV-A in China or any other part of the world, but it may be assumed that genetic exchanges had occurred when the HEV71 strain co-circulated with other HEV-A during that time period in China. In this study, we have already performed the Nucleotide acid and deduced amino acid sequence identities analysis, and Phylogenetic analysis based on 5′UTR, P1, P2, P3, 3′UTR region of the genome respectively. Both identities and phylogenetic analysis indicated that both C4a and C4b viruses had much higher sequence identities with EV71 in P1 region; while in the P2 and P3 regions, both C4a and C4b viruses had much higher sequence identities with CVA-16,14,4. Although we combined the simplot analysis, the identities and phylogenetic analysis to confirm the recombination of C4 EV71 from the prototype strains of CAV16, 14 and 4, it is still a hypothesized conclusion.
Species human enterovirus A (HEV-A) which include 11 members of the coxsackievirus A (CV-A) group; CVA2–8, CVA10, CVA12, CVA14, CVA16 and human enterovirus 71 (EV-71) are associated with several human diseases
In this study, we provided the evidence confirming that these recombination C4 viruses have been occurred in China since 1998 and persistently circulated in China more than 14 years, and evolved into 2 major evolution lineages, C4a and C4b viruses during the decade. More and more severe neurological diseases and fatal cases have been caused by the intertypic recombinant C4a viruses throughout mainland China since 2007. A total of 5,034,764 HFMD cases including 61,582 severe and 1,894 fatal cases were reported to NNDRS during 2008–2011 in mainland of China
This study did not involve human participants or human experimentation; the only human materials used were stool samples, throat swab samples, and vesicles collected from HFMD patients at the instigation of the Ministry of Health P. R. of China for public health purposes, and written informed consent for the use of their clinical samples was obtained from all patients involved in this study. This study was approved by the second session of the Ethics Review Committee of the Chinese Center for Disease Control and Prevention. The EV71 strains used in this study were isolated between 2002–2003 and 2007–2009 from stool, throat swabs, or vesicles from HFMD patients from different geographical locations in the Shanghai, Chongqing, Anhui, Shandong and Henan provinces of China (
Viral RNA was extracted from the viral isolates using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) and stored at -80°C until further use. The full-length genomes of 16 EV71 strains from the HFMD patients were amplified and sequenced. The viral RNA was converted to cDNA by a random priming strategy. The cDNA was amplified using the primers designed by multiple alignments of EV71 genomes available in GenBank database(
Sequencing analysis overlapping DNA sequences with at least 85% sequence homology and a minimum of 20 overlaps were assembled into contigs to generate consensus sequences using Sequencher version 4.0.5 (Gene Codes Corporation, USA). The consensus sequences were aligned against other EV71 complete genome sequences retrieved from the GenBank (
Two nucleotide alignments were generated using the MEGA 5.05
The 16 sequences reported in this study were deposited in the GenBank sequence database, accession numbers: EU703812 to EU703814; JX678874-JX678886.
List of 186 HEV71 strains.
(DOC)
Primers for RT-PCR, Sequencing and RACE.
(DOCX)
The difference between this study and other studies (ref. 19, 20, and 27) to identify different break point and parental strains of EV71 C4a and C4b recombinants.
(DOCX)
Phylogenetic trees showing the relationships amongst HEV-A isolates using the different genomic regions. The neighbour-joining trees were constructed from alignment of the 2A (a), 2B (b), 2C3A3B (c), and 3C3D (d) genomic region, respectively. The percentage of bootstrap (percentage of 1000 pseudoreplicate datasets) replicates supporting the trees are indicated at the nodes; for clarity, only values over 80% are shown. The branch lengths are proportional to the genetic distances corrected using Kimura-two-parameter substitution model.
(PPT)
We would also like to acknowledge all of the laboratories that isolated the viruses used in this study. And we thank anonymous reviewers for comments that improved the manuscript.